Detection of cartilage matrix degradation by autofluorescence lifetime

Abstract

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of noninvasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.

Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fiber-optic probe

“…Collagen type IV is primarily found in the linings surrounding the colonic crypts (basal membranes), whereas collagen types I-III are predominant in the stroma of the lamina propria [56,57]. Collagen fluorescence is particularly complex and a range of values are reported in the literature, e.g [58,59], and recent measurements of type II collagen obtained with an instrument similar to that used here showed a peak emission wavelength of 390-430 nm and a mean fluorescence lifetime of approximately 10 ns for excitation at 355 nm [60]. NADH has a peak emission wavelength of 460 nm and a mean fluorescence lifetime of 440 ps in its free state [61] and longer decay components typically in the range ~2-2.5 ns when protein bound [62].…”

Low Density Lipoprotein Receptor-related Protein 1 (LRP1)-mediated Endocytic Clearance of a Disintegrin and Metalloproteinase with Thrombospondin Motifs-4 (ADAMTS-4)

“…The anti-human ADAMTS-4 catalytic domain rabbit polyclonal antibody was raised in rabbit and characterized (9). Recombinant human ADAMTS-4, ADAMTS-5, their domain deletion mutants, MMP-1, MMP-13, and RAP were prepared as described previously (9,10,35,36). Recombinant human IL-1␣ was kindly provided by Prof. J. Saklatvala (Kennedy Institute of Rheumatology, Oxford, UK).…”

“…Quantitative Reverse Transcriptase-PCR-Quantitative reverse transcriptase-PCR was carried out as described previously (36). Briefly, cDNA was generated using a reverse-transcription kit (Applied Biosystems, Foster City, CA) and random primers from RNA were extracted and prepared using the RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer's guidelines.…”

A flexible wide‐field FLIM endoscope utilising blue excitation light for label‐free contrast of tissue

“…For this particular excitation wavelength, we expect to be able to measure fluorescence signals from the metabolite, flavo adenine dinucleotide (FAD) [27] and the intercellular matrix components, collagen [28,29] and elastin [27]. These endogenous fluorophores are considered to be potential biomarkers for disease progression [30].…”